Isolation of mutations defining five new cistrons essential for development of bacteriophage Mu

VIROLOGY

93, 303-319 (1979)

isolation

of Mutations Defining Five New Cistrons Development of Bacteriophage Mu

MARTHA M. HOWE,’ KATHRYN Department of Bacteriology,

Essential

for

J. O’DAY,2 AND DENNIS W. SCHULTZ

University

Madison, Wisconsin 53706

of Wisconsin,

Accepted November

8, 1978

Three hundred new mutant strains of bacteriophage Mu with amber mutations in essential genes were isolated and characterized. Measurement of complementation between these new strains and strains carrying mutations in previously identified complementation groups revealed the existence of five new complementation groups: T, U, V, W, and Y. In general, mixed infection of cells by phage carrying mutations in different cistrons resulted in the production of a normal burst of 50 to 200 phage per cell; however, mixed infection with phage carrying mutations in certain pairs of adjacent cistrons gave a loto 20-fold lower burst of only 3 to 30 phage per cell. Cistron pairs which showed this reduced complementation were D-E, H-F, J-K, Y-N, Q-V, W-R, and S-U. In addition, mutations assigned to cistron I fell into three groups on the basis of their complementation behavior; phage carrying mutations in one group located at one end of the cistron showed weak complementation with phage carrying mutations in a second group at the opposite end of the cistron, but phage carrying mutations in the third group located between the first two groups showed no complementation with members of any of the three groups. The complementation analysis also showed that mutations previously assigned to separate complementation groups 0 and P really belong to a single complementation group P. INTRODUCTION

Recently during an extensive search for integration-deficient mutants of Mu (O’Day In the early 1970s several laboratories et al., 1978) we isolated a large number of isolated amber mutations in genes essential new strains containing amber mutations in for the development of bacteriophage Mu, assigned the mutations to specific cistrons essential genes of Mu. Since it would be useon the basis of complementation tests, and ful to have a more extensive set of mutations mapped the mutations by assaying rescue of and more precisely defined map for analysis the wild-type allele from bacterial strains of Mu gene organization, function, and regucontaining partially deleted Mu prophages lation, we have undertaken complementa(Boram and Abelson, 1973; Bukhari and tion and mapping analysis of some of these Metlay, 1973; Faelen and Toussaint, 1973; mutant strains. The results of complementation tests Howe, 1973a; Wijffelman et al., 1973). The have allowed us to define five new cistrons integration of these sets of mutations and maps into a single comprehensive map con- essential for Mu development, and the maptaining 20 essential cistrons defined by 59 ping analysis (O’Day et al., 1979) has remutations was accomplished during the Mu sulted in the production of a precise and Workshop held at Cold Spring Harbor detailed genetic map of mutations in these Laboratory in July of 1972 (Abelson et cistrons. al. 1973). 1 Author to whom reprint requests should be addressed. 2 Present address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, Mass. 02111. 303

MATERIALS

Bacterial

AND METHODS

and Bacteriophage

Strains

Bacterial strains. The bacterial strains used are listed in Table 1. 0042-6822/79/040303-17$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

HOWE, O’DAY, AND SCHULTZ

304

TABLE 1 BACTERIAL STRAINS Genotype”

Strain

Source/Reference/Derivation

159 CA5013 CSH25 D5009

F- gal uvr strA HfrH lacy SuI+ F- SuIII+ pro Hfr Cav. thr leu met lac ara mtl xyl strA

KD1067 KMBL1644 Ml07 MH394 MH594 MH1212 MH2500 MH2502 MH2503 MH2504 MH2505

F- arg his m&D Su+ b HfrH thi met pA(tonB-trp::+MuAcA) F-Alac SuI+ strA F-Apro-lac his met tyr strA F-Apro-lac his met tyr &A NalR F- SuI+ uvr leu &A’ F- gal strA recA F- SuI+ uvr leu strA AR F- SuII+ thr leu tonA lac AH F- SuIII+ XRpro F-Apro-lac his met tyr NalR AK F-arg his m&D Su+ (Mu cts62) F-arg his mutD Su+ (Mucts62) (AKam24) F- SuII+ thr leu tonA lac

tonA tsz azi

MH2509

MH2510 $5003 WD5021

F- SuIII+ me1 pro F- gal &A lac

Hendrix (1971) E. Signer Cold Spring Harbor Collection E. Signer Degnen and Cox (1974) Wijffelman et al. (1973) Howe (1973a) 533 of Zeldis et al. (1973) MH394 NalR M. Howe WD5021 recA F. van Vliet MH1212 AR QIAR CSH25 AR MH594 AR

KD1067 (Mu cts62) MH2509 (AKam24) Howe (1973a) Howe (1973a) Howe (1973a)

n The ::Mu notation indicates that the Mu prophage is integrated in the gene immediately preceding the ::. The +Mu and -Mu notation designates the orientation of the prophage as described in Howe and Bade (1975). The pA( tonB-trp:: +MuAcA) notation indicates a partial deletion of the region within the parentheses; in this case the deletion removes at least part of tonB and the immunity and A cistrons of Mu inserted in tv. * This suppressor has not been characterized, but is probably the same suppressor as that in strain AB1157. c Strain MH1212 was derived from strain 159 in two steps. First strain 159 was crossed with strain D5009 and a gal uvr leu &A recombinant was isolated and designated WC5010. This strain was then mated with strain CA5013 and a uvr leu strA SuI+ recombinant was isolated and designated MH1212.

Bacteriophage strains. Mu c+ and its heat-inducible derivative, Mu cts62, have been described previously (Howe, 1973a). Mu ~25 is a phage containing a spontaneous clear mutation.Mu phage with amber mutations 1001 to 1256 were isolated and partially characterized by Howe (1973a). The isolation of phage with mutations 1257 to 1999 and 7000 to 7367 is described in this report. Mu phage with amber mutations in the 2000 series were from C. Wijffelman et al. (1973); the 3000 series was from M. Faelen and A. Toussaint (1973); the 4000 series was from W. Boram and J. Abelson (1973); and the 5000 series was from T. Razzaki and A. Bukhari (1975). Mu vir (Mu vir3057 mom) was obtained from A. Toussaint (van Vliet et al., 1978). hKsus24, Avir,

and P2virl were obtained from W. Dove, E. Signer, and M. Sunshine, respectively. Media

LB broth, LB agar, soft agar, and SB have been described previously (Howe, 1973a). LBM and SBM contain 2 x lo+’ M MgSO, in addition to LB and SB. LBMC is LB containing 2 x low3 M MgSOs and 2 x 10m3M CaCl,. SBPM is SB containing 1 x lop3 M Pb(C2H30& and 2 x 10e3M MgSO,. SM contains per liter 5 g NaCl, 2.7 g Tris-HCl, and 0.32 g Trizma Base and is supplemented with MgS04 to a iinal concentration of 2 x 10e3M after autoclaving. SMC is SM containing 2 x 10m3M CaCl,. TCM contains 0.01 M Tris pH 7.5, 0.01 M

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MU

305

MgS04, and 0.01 M CaCl,. Minimal medium Mall (white) colonies were cross-streaked is OM of Ozeki (1959) supplemented after against Avir. Both Mal+ and Mal-h resistant autoclaving with vitamin B, at 1 pug/ml, cells were isolated. sugars at 0.2%, and desired amino acids at 20 pg/ml. EMB is that of Luria et al. (1960). Mutagenesis

Bacteriophage

techniques

Mu lysate preparation by lytic infection [modi,fied from Howe (1973a)l. Cells were

procedures

Hydroxylamine (HA) mutagenesis of free phage (Howe, 1973a). The following

solutions were mixed and incubated at 37”: 1 ml of 1.3 M hydroxylamine hydrochloride grown in LBM to 2 x lo* cells/ml at 3’7”. pH 6.0, 1 ml of 0.5 M NaH,PO,*H,O adOne fresh plaque was picked with a Pasteur justed to pH 6.0 with NaOH, 0.5 ml of 0.04 pipet and added to 0.3 ml of cells, vortexed, M (0.08 M or 0.16 M) Na,EDTA, and 0.15 incubated at 3’7”for 15 min, and diluted with ml of Mu c+ lysate. Afterward, 13- and 6.0 ml of LBM at 37”. The culture was 24-hr samples were removed and diluted grown at 37” until lysis (3-5 hr). If the loo-fold into SMC supplemented with lop3 cells became more concentrated than log/ml, M Na,EDTA. Dilutions were plated on they were diluted threefold with fresh mixed indicator plates to determine the LBM. After lysis, the lysates were chilled, degree of phage survival (7-30%) and the chloroform was added, and debris was re- frequency of amber mutations (0.3-1.0%). moved by centrifugation. (Sorvall SS-34 This procedure produced mutants numrotor for 30 min at 12,000 rpm.) This pro- bered 7021-7099, 7103-7105, 7108-7135, cedure generally resulted in titers of 108- 7153. lOlo plaque-forming units (PFU)/ml; howMutagenesis of phage by growth in a ever, with some amber mutant phages it mutator strain of E. coli KD1067 (mutD) produced titers of only lo7 PFU/ml. (Degnen and Cox, 1974). Mu lysate preparation by induction of a (a) Low multiplicity lytic infection: Strain lysogen. Lysogens were isolated as de- KD1067 was grown in OM minimal medium scribed previously (Howe, 1973a,b). A heat- supplemented with glucose, arginine, and inducible lysogen was grown in SBPM to histidine to 2 x lo* cells/ml, diluted fivefold 2 x 108-4 x lOacells/ml at 32 or 34”. Phage with LBM, and again grown to 2 x lOseells/ development was induced by adding an ml. One fresh Mu c+ plaque was added to equal volume of medium at 55” and incubat- 0.5 ml of cells, incubated for 15 min at 37” ing the culture at 42”. After 20 min the cul- and diluted with 7.5 ml of LBM at 37”. The ture was shifted to 37” until lysis. Chloro- infected cells were grown at 37” until lysis. form was added, and debris was removed The lysate was chilled, chloroform was by centrifugation. (Titers: 1 x log-2 added, and debris was removed by centrifx lOlo PFU/ml.) ugation. (Titers: 1 x lo*-3 x log PFUl Titration of Mu lysates. Indicator cells ml.) This procedure gave 0.3-0.5% ambers were grown in LB to log cells/ml. Phage and mutants numbered 1264, 1281, 1270, were diluted in SMC. Portions of 0.05 to 1294, 1304, and 1308. (b) High multiplicity lytic infection: 0.2-ml diluted phage and 0.1 ml of cells were mixed and incubated at 37” for 20 min. Soft Strain KD1067 was grown in minimal medium to 2 x lo* cells/ml, diluted fivefold agar, 2.5 ml, was added, and the mixture was plated on thick LB plates. The plates with LBM, and again grown to 2 x 10’ were incubated overnight at 37” (occa- cells/ml. Five milliliters of cells was centrifuged and resuspended in 5 ml of a Mu c+ sionally at 32 or 42”). Isolation of h resistant mutants. Xvir was lysate previously grown on strain KD1067 spotted on a lawn of cells on an LB plate in LBM to give a multiplicity of infection and incubated overnight at 37”. Colonies (m.o.i.) of 4. The infected cells were incuwhich grew in the clear spot were purified bated with aeration at 37” until lysis. The on EMB maltose plates. Mal+ (red) and lysate was chilled, chloroform was added,

306

HOWE,

O’DAY,

and debris was removed by centrifugation. (Titer: 5 x lOa PFU/ml.) This procedure gave 0.3% ambers and mutant 1300. (c) Heat induction of a Mu cts62 lysogen: Strain MH2509 (KD1067 Mu cts62) was grown in SBPM to 7 x lo* cells/ml at 32”. An equal volume of medium at 55” was added, and the cultures were grown at 42” until lysis. The lysate was chilled, chloroform was added, and debris was removed by centrifugation. (Titer: lo7 PFU/ml.) This procedure gave l-2% ambers and produced mutants numbered 1504-1564 and 7000-7003. (d) Plate lysate: Strain KD1067 was grown in LB to lo9 cells/ml. Phage, O.l0.9 ml, derived by suspending one fresh Mu C+ plaque in 1.0 ml of LBCM was added to 5 x 10’ to 2 x lo* cells and incubated for 30 min at 32”. Soft agar, 2.5 ml, supplemented with 5 x lop3 M MgS04 and 5 x low3 M CaCl, was added to each tube, and the mixtures were poured onto LB plates. The plates were incubated at 37” until lysis was confluent. LBCM broth, 2.5 ml, was added, and each plate was reincubated for 1 hr. LBM, 3 ml, was added, and the broth and agar overlays were transferred to tubes. Chloroform was added, and the agar and debris were removed by centrifugation (Sorvall SS-34 rotor for 20 min at 7000 rpm). Some phage which were trapped in the agar were eluted by mixing the pellet with 3 ml of LBM and again removing debris by centrifugation. The two supernatants for each lysate were combined and stored at 4°C. (Titers: 2 x 106-5 x lo6 PFU/ml.) This procedure gave 0.6-0.9% ambers and mutants numbered 1690 to 1917. (e) Attempted enrichment for mutations in early functions: Since we were particularly interested in isolating mutations in early functions of Mu, we designed a procedure which would enrich for such mutations by virtue of the relative heat resistance they would confer upon a Mu cts lysogen (Howe, unpublished observations). The basic rationale of the procedure was to enrich for heat-resistant lysogens of the Su- mutD strain and then to recover the expected A-defective and B-defective phage by inducing Mu development in the

AND SCHULTZ

presence of a superinfecting hpMu phage which could supply Mu A and B functions (Howe, unpublished observations). Heat selection. A culture of strain MH2510 [KDl067 (Mu cts62) (hKsus24)] was mutagenized by growth at 32” in 5 ml of SBM to lo* cells/ml. Anti-Mu antiserum, 0.1 ml (k - 100 min-‘) (Howe, 1973a) was added to the culture which was shifted to 42” for 1 hr and then grown overnight at 32”. The procedure was repeated once using the overnight culture as an inoculum. Infection of the survivors with ipMu 377. The heat-treated culture was grown to 2 x lo* cells/ml in SBM, washed twice with SBM by centrifugation to remove antiserum, and infected with ApMu 377 (which carries Mu genes c, A, and B) at an m.o.i. of 5 for 15 min at 32”. Five milliliters of SBM was added, and the culture was grown at 42” for 180 min. Chloroform was added and debris was removed by centrifugation. A second enrichment was done with the following changes in procedure: The heattreated culture was grown in SBM supplemented with 0.02% maltose prior to infection with ApMu 377 to induce the A receptor sites. The ApMu-infected cells were induced at 42” for only 90 min. The lysates were titered on mixtures of Su+ and Su- A-resistant indicator cells (MH2502-MH2505), and amber mutant phage were identified by their plaque morphology. (Titers: 5 x 105-1 x lo* PFU/ml.) This procedure gave 0.5-1.0% ambers and mutants numbered 1920 to 1998, 7006 to 7020, 7100, 7106 and 7140 to 7367. Most of the defective phage recovered from this procedure contained mutations in late Mu genes. Only seven phage defective in early Mu functions were recovered; one phage contained a mutation in the A gene (7302) and six phage contained mutations in gene B (1979, 7076, 7154, 7234, 7308, and 7342). The mutD strain MH2510 lKD1067 (Mu cts62) (AKsus24)] used in this procedure was later found to carry an unidentified amber suppressor. In retrospect, it is not surprising that the enrichment procedure was not very successful, since suppression of the early gene mutations would

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abolish the selective advantage of the mutant strains. All of the mutations in genes A and B isolated by this technique are suppressed by the KD1067 suppressor; therefore it seems unlikely that their presence in the lysates was due to any enrichment by the heat treatment. Mixed Indicator for Isolation and Scoring of Phage with Amber Mutations

Su- (MH594) and Su+ indicator cells were grown in LB to 5 x lOa cells/ml. Phage, 0.1 ml, diluted in SMC, was mixed with 0.1 ml of Su+ indicator cells. After 20min adsorption at 37”, 0.1 ml of Su- cells, previously diluted two- to five-fold in LB, was added and the mixture plated in 2.5 ml of soft agar on thick LB plates. The plates were incubated overnight at 37”. On these plates wild-type plaques had sharply defined clear edges and turbid centers, while amber mutant plaques had fuzzy edges and were uniformly turbid. In addition, amber mutant plaques were often much smaller than wild-type plaques. The ratio of Su+/ Su- cells was varied from 2 to 5 in order to isolate phage with both leaky and nonleaky amber mutations. SuI+ (MH1212), SuII+ (Ql), and SuIII+ (CSH25) indicator cells were used in order to enable the isolation of phage with amber mutations which required a specific suppressor. Phage suspected of having amber mutations were picked with a toothpick and transferred to lawns of Su- and Su+ indicator cells. Phage which grew on the Su+ indicator but not on the Su- cells were kept for further testing. All the amber mutations characterized were isolated in this manner. Complementation

Tests

On plates. Su- recA (MH2500) cells were grown in LBMC to log cells/ml. Amber mutant lysate, 0.1 ml, at lo8 to log PFU/ml was mixed with 0.3 ml of cells, incubated for 15 min at 37”, and plated in soft agar on LB plates. Lysates were spotted onto the plates and onto control plates containing uninfected Su- recA cells. The plates were incubated overnight at 37”. If lysis occurred

MU

307

in a doubly infected spot and little or no lysis occurred in the singly infected controls, it was concluded that the two phage could complement each other. Each mutation was originally tested with one member of each known complementation group: A1093, B1066, C4005, lys1025, 03801, E1006, F1065, G1042, H1043,14037, J1155, KlOlO,L1007, M1114, N1041,01133, P1024, Q1074, R1059, S1004. Placement of a mutation in a particular complementation group was confirmed by complementation tests between the mutant phage and one or two additional phage carrying mutations in that group: A3011, B1032, B5179, C2005, lys2009, E2003, F2008, F3007, G1021, G3002, H3009,11077,

12021, J2002, K2051, L1033, L2010, M1124, NlOOl, N(now Y)1027, P1008, P1012, R(now W>lOll, R(now W)1044, U1049, U1050, S1063. In liquid. Su- recA (MH2500) cells were grown to lo8 cells/ml in SBPM, centrifuged, and resuspended in 0.2 vol. of TCM, a buffer which facilitates adsorption of Mu. Cells, 0.1 ml, were mixed with 0.1 ml of two amber mutant lysates to give an m.o.i. of 4 to 10 for each phage. After I5 min of adsorption at 37”, 0.1 ml diluted anti-Mu antiserum (final k - 1 min-l) was added to inactivate unadsorbed phage, and the mixture was incubated for 10 min at 37”. The mixture was then diluted loo-fold in SBM and incubated with aeration at 37” for 60 min. The cultures were treated with chloroform and titered on appropriate Su+ and Su- recA indicator cells. As controls infections were done with each of the amber mutant lysates alone and with a complementing amber mutant lysate. In some experiments unadsorbed phage (usually 2-5% of input) were measured by removing an aliquot after 15 min of adsorption, diluting in SMC containing chloroform, and titering on Su+ indicator cells. In most cases interpretation of the complementation test results was straightforward. Mutations were assigned to separate complementation groups when the mixed infection produced a normal burst of 50 to 200 phage per cell. Mutations were assigned to the same complementation group when

308

HOWE,

O’DAY,

AND SCHULTZ

the burst from the mixed infection was no cells in the lawns were varied so that higher than the bursts from the single in- mutants differing greatly in their growth fections. In cases where the phage bursts characteristics could still be detected. This were at intermediate levels, the phage approach was used in an attempt to minimutant pairs were considered to belong to mize loss of specific classes of mutants due separate complementation groups if the to their potential poor growth in Su+ or mixed infection produced a burst of at least relatively leaky growth in Su- cells. Success 2 phage per cell and if that burst was at in this regard was indicated by the isolation, least lo-fold higher than the burst pro- at a frequency of approximately 5% each, duced in each single infection. A few rare of mutants too leaky in Su- to be characterexceptions to the above limits were made ized and mutants too defective in Su+ to be for mutant phage which showed generally grown and analyzed. Approximately 300 new amber mutant poor complementation or those which gave burst sizes greater than 1 phage per cell phage were characterized by complementain single infection. tion and marker rescue analysis. At least 83% of these mutant phage arose independently since they were obtained from difRESULTS ferent mutagenized lysates or behaved differently in complementation or marker Isolation of Amber Mutants rescue assays. In order to increase the frequency of mutants in the phage populations to be Complementation Analysis of Amber examined, Mu phage were mutagenized Mutations either by treatment of lysates with hydroxylamine, a chemical mutagen which causes Preliminary assignment of mutations to unidirectional transitions (Tessman et al., complementation groups was made on the 1964), or by growing the phage in E. coli basis of results of spot complementation strains containing the mutator allele m&D. tests performed on lawns of recA Su- cells. The m&D is a conditional mutator allele In these tests each new mutant phage was which causesbidirectional transitions, trans- tested for complementation with one to versions, frameshifts, and deletions when three representative mutant phage defecthe cells are grown in rich media (Fowler tive in each of the 20 known complementaet al., 1974). In this work extensive use tion groups, A through S and lys. The was made of mutD mutagenesis because it majority of the mutants could be unambiguproduced a high frequency of mutants and ously assigned to known complementation because it allowed the phage to be grown, groups because phage containing them mutagenized, and recovered in a single could complement with phage defective in step. Several different mutagenesis pro- all complementation groups except one. In cedures were used in an attempt to maximize these tests phage defective in a new group the efficiency of mutagenesis which was T were detected by their ability to commonitored by measuring the frequency of plement phage defective in each of the phage containing amber mutations in previously defined complementation groups. essential genes. Details of the procedures, The tests also revealed that phage defective the resulting amber mutant frequencies, in 0 were also defective in P and vice versa. In the spot tests a number of mutant and the specific mutants arising from each procedure are given under Materials and phage behaved anomalously in that they Methods. appeared completely or partially negative The amber mutants were detected by for complementation with phage defective their fuzzy and uniformly turbid plaque in two adjacent complementation groups. morphology in lawns containing a mixture To prevent mistakes in the assignment of of permissive Su+ and nonpermissive Su- mutations to complementation groups, cells. The relative amounts of Su+ and Su- selected mutants were tested using a more

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quantitative assay for complementation. This assay involved measuring the phage burst produced after a single cycle of mixed infection of Su- recA cells in liquid. Such assays were performed with phage mutant pairs expected to be in the same complementation group and pairs expected to be in adjacent complementation groups. Results from these burst assays not only allowed the unambiguous assignment of mutations to complementation groups but also revealed the existence of four additional complementation groups which were not detectable in the spot test analysis. The results of these complementation tests are described below for each complementation group. Group A. Spot complementation tests indicated that five new mutant phage were defective for cistron A. This assignment was confirmed by results of burst assays in which mixed infection of phage containing mutation A7110 with those containing mutations A1504, A3011, and A1093 produced less than 0.1 phage per cell while control infections of each A amber phage with phage carrying mutation V7165 produced 23-75 phage per cell. With the exception of phage carrying mutation A7110, each of the A amber mutant phage was defective in integration in an Su- host (O’Day et al., 1978). Phage carrying mutaTABLE

MU

309

tion A7110, which is located near the promotor-distal end of the cistron (O’Day et al., 1979), showed low levels of integration @‘Day et al., 1978). Croup B. Six new mutant phage were found to be defective in cistron B. Results of burst assays, presented in Table 2, showed that mixed infections of B-defective phage produced less than 1 phage per cell while control mixed infections of B and R-defective phage produced 36 to 112 phage per cell. The B mutations examined included mutations 5150 and 5175 to 5179, which were isolated by A. Bukhari as “BU” amber mutations which prevented the cell death normally associated with heat treatment of a Mu cts lysogenic strain (Bukhari, 1975; Razzaki and Bukhari, 1975). These mutations mapped among B mutations (O’Day et al., 1979) and behaved like B mutations in both spot and liquid burst complementation assays (Table 2). Mutations 5175 (Table 2) and 5176 (data not shown) were anomalous in that phage carrying them were also partially defective in complementation with phage carrying A mutations. Group C. Only one new mutation in the C cistron, C1966, was found. Phage containing the Cl966 mutation gave less than 1 phage per cell in mixed infection with phage carrying the C2005 mutation while both C-defective phage gave bursts of 60 to 2

COMPLEMENTATION OF A AND B MUTANT PHAGE” Burst sizes Mutants

A1093

A3011

B7154

B5150

B5175

B5177

B1066

A 1093 A3011 B7154 B5150 B5175 B5177 B1066 R1059

0.08 0.18 39 34 1.2 15 21 59

0.36 44 22 0.7 21 18 94

0.04 0.02 0.18 0.22 0.02 112

0.02 0.16 0.22 0.04 100

0.30 0.76 0.16 44

0.74 0.18 53

0.02 36

a Complementation tests were performed by mixed infection of amber mutant phage in recA Su- cells in liquid as described under Materials and Methods. The results are presented as the burst sizes (phage per cell) of Mu amber phage produced by the infected cells. The burst size of the control phage mutant R 1059 in single infection varied from 0.05 to 0.15 in different experiments.

310

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80 phage per cell in mixed’infection with phage carrying mutation W1044. Group lys. Seven new mutations were easily assigned to the lys cistron on the basis of results of spot complementation tests. Burst assays of complementation of phage carrying the lys1030 mutation with those carrying lys7297,lys7258, and lys1025 gave less than 1 phage per cell while control complementations of lys-defective phage with phage carrying an H 7222 or W 1044 mutation gave 80-180 phage per cell, thus confirming the assignment of the Zys mutations to a single cistron. Groups D and E. Mutations in these groups could not be unambiguously assigned to either the D or E complementation groups on the basis of spot tests. Phage with mutations in subgroup 1 (1888, 7112, 7147, 7163, 7291, 7293, 7310) did not complement phage with any of the D or E mutations. Phage with mutations in subgroup 2 (7015, 7031, 7088, 7330) showed very weak complementation with phage carrying the E2003 mutation but not the 03801, E3005, or El006 mutations. Phage with mutations in subgroup 3 (1270, 1841, 7040, 7045, 7073, 7114, 7116, 7148, 7192, 7288, 7295, 7312, 7329, 7344, 7346, 7367) showed very weak complementation with phage carrying the E3801 mutation but not the E2003, E3005, or E 1006 mutations. The results of the burst assays shown in Table 3 clearly demonstrate that these mutations comprise two distinct eomplementation TABLE

groups, D (subgroup 2) and E (subgroups 1 and 3). Mixed infections with phage carrying mutations in the same group produced bursts approximately the same as infections with only one mutant phage type (0.005 to 2.5 phage per cell depending on the leakiness of the specific mutations used), whereas mixed infections with phage mutant in different groups produced bursts which were significantly higher (3 to 50 phage per cell) but still not as high as those from mixed infections with phage carrying a more distant R mutation (38 to 125 phage per cell). The relatively low degree of complementation observed between phage with D and E mutations is consistent with their ambiguous behavior in the spot tests and suggests that bursts of 1 to 15 phage per cell measured in liquid may not be high enough to produce lysis in the spot test. Groups H and F. The results of spot complementation tests allowed mutations in these groups to be placed in one of three subgroups: Seventeen phage were defective for F but not H, 5 were defective for H but not F, and 27 were defective for both H and F. Burst assays such as those shown in Table 4 revealed that phage defective for both H and F in the spot tests (e.g., 7100 and 7222) did not complement each other or phage carrying H mutations but did complement phage carrying F mutations to produce 10 to 20% of the normal burst. On this basis the mutations defective in 3

COMPLEMENTATIONOF D AND E MUTANTPHAGE" Burst sizes Mutants

07330 07088 01031 E7291

07330

07088

01031

E7291

0.5 3 5 6 6 50

0.005 0.006 0.02 0.02 73

E7293

E7288

E3005

0.03 0.04 56

0.006 38

0.07 1.25 0.08

1.0 1.0 10 11 11

E7293 E7288 E3005

23 26 24 53

29

R1059

125

125

” See legend for Table 2.

0.01

0.02 0.03 73

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TABLE

MU

311

4

COMPLEMENTATIONOFH AND F MUTANTPHAGE~ Burst sizes

Mutants H1932 H1519 H7100

H7222 F7217 F7207 R 1059

H1932 3.9 7.4 1.2 1.8

94 79 68

H1519

H’7100

H7222

F7217

F7207

9.4 5.1 4.5 75 106 164

0.01 0.03 22 24 113

0.01 21 21 129

0.32 0.31 153

0.32 153

a See legend for Table 2.

H and F and those defective only in H in spot tests have been classified as H mutations while those defective only in F have been assigned to the F cistron. It should be noted that almost all the H mutations showed the partially defective F phenotype; the only exceptions among the newly isolated mutations were 1519, 1932, 1996, 7000, and 7333 which were all located at the promotordistal end of the cistron (O’Day et al., 1979) and appeared to be particularly leaky mutations. Group G. Twelve new phage defective in cistron G were easily detected from results of spot complementation tests. Contiming burst assays gave less than 1 phage per cell for mixed infections of phage carrying mutation G1056 with those carrying mutations G7177, G7360, and G1021, while control mixed infections of G-defective phage with phage carrying mutations H 7222 or W 1044 gave 40 to 60 phage per cell. Group I. In spot complementation tests 28 new mutant phage were found to be defective for complementation with phage carrying Z mutations. However, when tested by liquid burst assays, each phage fell into one of three subgroups. Phage containing mutations in the first subgroup (mutations 3026, 4037, 7011, 7150, 7155, 7247, 1991, 1053, 1952, 2021, 7035, 7146, 7340, 1516, 1862, 1994, 1961, 1993, 7002, 7003, 7160, 7196, and 7281) showed no complementation (co.1 phage per cell) with phage carrying mutation 11991, a member of subgroup 1, but showed 5-20% of the

normal level of complementation (3 to 15 phage per cell) with phage carrying mutation 11077, a member of subgroup 3. Phage containing mutations in subgroup 3 (1077, 7010, 7069, 7057, 7149) showed an opposite pattern giving no complementation with phage carrying mutation 11077 (subgroup 3) but 5-15% normal complementation with phage carrying mutation 11991 (subgroup 1). Phage carrying mutations in subgroup 2 (1525, 1973, 7280, 7349, 7350) gave no complementation (co.2 phage per cell) with either phage carrying 11991 or 11077. In all cases the control infections of I-defective phage with phage carrying the N1041 mutation gave large bursts, usually 40-200 phage per cell. An example of the complementation behavior of phage with these mutations is shown in Table 5. Mapping of these mutations (O’Day et al., 1979) has revealed that mutations in each subgroup map as a contiguous group with mutations in subgroup 2 mapping between mutations in subgroups 1 and 3. At present, due to the existence of subgroup 2 mutations, all these mutations have been assigned to a single cistron, I; however, it is quite possible that future experiments will indicate that mutations in subgroups 1 and 3 should be assigned to separate cistrons (subgroup 3 would then become cistron 2 >. Group T. Phage with amber mutations 1913 and 7327 did not complement each other but did complement phage with representative mutations in all other complementation groups in spot tests. Since these

HOWE,

312

O’DAY,

AND SCHULTZ

TABLE

5

COMPLEMENTATIONOFI MUTANTPHAGE~ Burst sizes Mutants

17155

11991

11994

11525

17350

I1077

I7149

I7155 11991 11994 11525 17350 11077 17149 V7165

0.04 0.03 0.02 0.12 0.06 5.3 2.9 118

0.02 0.02 0.12 0.08 5.2 2.5 100

0.02 0.13 0.05 9.4 2.0 153

0.15 0.10 0.06 0.08 100

0.08 0.04 0.06 76

0.04 0.02 65

0.02 88

a See legend for Table 2. Mutations 7155, 1991, and 1994 belong to subgroup 1, mutations 1525 and 7350 belong to subgroup 2, and mutations 1077 and 7149 belong to subgroup 3. Infection of phage V7165 alone produceda burst-of 0.15 phage per cell.

mutations were located between I and J mutations (O’Day et al., 19’79),burst assay complementation tests were performed with phage mutant in the I-J region. As shown in Table 6, phage carrying mutation 7327 complemented well with phage defective in genes G, I, J, or K. In a similar test phage carrying mutation 1913 did not complement those with mutation 7327 (burst size ~0.01 phage per cell) whereas they complemented well with phage mutant in genes G, I, J, or K (40-140 phage per cell). Therefore, these two mutations define a new complementation group T. Groups J and K. In spot complementation tests phage with new mutations in these groups fell into one of three classes: Three TABLE

(7018, 7182, 7298) failed to complement J-defective phage, but did complement K-defective phage, and were therefore assigned to the J cistron, eight complemented J-defective phage but not Kdefective phage and were assigned to the K cistron, and four new (1819, 1963, 7076, 7130) plus one old (1005) failed to complement either J or K-defective phage. Liquid burst assays (Table 6) revealed that phage carrying the J1005 mutation gave no complementation with phage carrying a J mutation and weak complementation with phage carrying a K mutation. Therefore, the mutations conferring a J-defective, partially K-defective phenotype have been assigned to the J cistron. This assignment 6

COMPLEMENTATIONOFG,I, T, J, K MUTANTPHAGE" Burst sizes Mutants

G1056

14037

I1077

T7327

J1005

J1155

KlOlO

G1056 14037 11077

0.3 71 57 77 105 140 129

0.06 0.9 17 27 51 37

0.01 49 93 92 117

0.01 43 86 44

0.23 0.29 4.0

0.26 17

0.06

T7327 J1005

J1155 KlOlO

a See legend for Table 2.

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was confirmed by mapping analysis (O’Day 1979) which indicated that the J mutations which were partially K defective were located early in the J cistron before J mutations which were not defective in cistron K. Group L. Nine new mutations were assigned to the L cistron on the basis of spot tests. Confirming burst assays showed no complementation (co.01 phage per cell) between phage carrying L1007 and L7156 mutations and normal complementation (40-100) phage per cell between the Ldefective phage and a phage carrying the H 7222 mutation. Group M. Six new mutations were assigned to the M cistron from their behavior in spot tests. This assignment was confirmed by burst assays which gave ~0.1 phage per cell from mixed infections of Ml954 and M 7251, M 1954 with M 7174, and M 7251 with M 7016. Bursts of 30-100 phage per cell were obtained from mixed infections of each M-defective phage with phage carrying mutation H 7222 or W 1044. Groups Y and N. Thirty-nine new mutant phage appeared to be defective in cistron N in spot complementation tests; however, burst assays revealed that these mutant phage actually fell into two complementation groups. Twenty-seven of these phage were tested by burst assay for complementation with phage carrying mutations 1027 and 1041. The phage clearly fell into two groups: those designated Y which showed no complementation with phage et al.,

MU

313

mutant 1027 and weak complementation with phage mutant 1041, and those designated N which showed no complementation with phage mutant 1041 and weak complementation with phage mutant 1027. An example of the complementation behavior of these mutant phage is shown in Table 7. Mapping analysis (O’Day et al., 1979) of these mutations demonstrated that all the Y mutations clustered in a contiguous group located to the left of all the N mutations. Group P. Phage carrying 14 new mutations in this group did not complement phage with either 0 or P amber mutations in spot tests. Furthermore, a control experiment revealed that phage containing 01133, which was originally assigned to a separate complementation group on the basis of spot tests on Su- Ret+ strains (Howe, 1973a), did not complement phage with P1008, P1012, or P1024 mutations in spot tests on the Su- recA strain. In liquid burst assays phage with P1024, P1008, and P1012 mutations gave burst sizes of
TABLE

7

COMPLEMENTATION OF Y AND N MUTANT PHAGE~ Burst sizes Mutants

Y1027

YlOOl

Y1018

N1041

N’7184

N1988

Y1027 YlOOl Y1018 N1041 N7184 N1988 V7165

0.04 0.02 0.03 24 34 25 118

0.01 0.02 3.2 15 3.5 100

0.02 8.8 29 12 124

0.02 0.04 0.05 124

0.04 0.06 124

0.05 106

m See legend for Table 2. Infection

of phage V7165 alone produced a burst of 0.02 phage per cell.

314

HOWE,

O’DAY,

designated P. The mistaken assignment of the 1133 mutation to a separate cistron in earlier studies was probably due to the formation of large numbers of recombinants between 1133- and P-defective phage in the Su- Ret+ host due to the relative leakiness of the 1133 mutation. Groups Q and V. Five new mutant phage appeared to be defective in cistron Q in spot test; however, burst assays testing complementation with phage carrying mutations 7165 and 1074 revealed that these phage actually fell into two distinct complementation groups Q and V. Phage carrying mutations Q1976 and Q1074 gave bursts of co.1 phage per cell in single or mixed infection with each other. Similarly, phage with mutations V7165 and V7213 gave bursts of co.02 phage per cell in single or mixed infection. Control mixed infections of each phage with phage carrying the R1059 mutation produced burst sizes of 147 to 200 phage per cell. Mixed infections of each Q mutant phage with each V mutant phage produced burst sizes of 2.9 to 11 phage per cell. On the basis of this low but significant complementation the mutations were assigned to separate complementation groups, Q and V. Groups W and R. Twenty-four new mutant phage appeared defective in cistron R in spot tests, but were resolved into two cistrons, R and W, on the basis of burst assays. Each of these mutant phage were tested by burst assay for complementation with phage carrying mutations 1044 and 1998. The phage fell into two distinct groups which gave no complementation with one mutant phage and low (5-20 phage per cell) complementation with the second mutant phage. Phage carrying W mutations 1011 and 1044 gave bursts of ~0.1 phage per cell in single and mixed infection with each other. Phage carrying R mutations 1028, 1059, and 7212 also gave bursts of co.1 phage per cell in single and mixed infection with each other. Mixed infections of each W with each R mutant phage produced bursts of 12 to 34 phage per cell, while mixed infections of W or R mutant phage with phage carrying the S1063 mutation produced bursts of 35 to 77 phage per

AND SCHULTZ

cell. Mapping analysis (O’Day et al., 1979) revealed that all mutations in the W cistron were located to the left of all mutations assigned to cistron R. Groups S and U. Phage carrying four mutations (1004, 1063, 1049, and 1050) which did not complement each other in spot tests were used as the standard phage to define the S complementation group. Spot complementation tests with phage carrying new mutations gave ambiguous results in that many of the phage did not complement any of the phage with the four standard mutations; however, a small set of phage with mutations 1294,1802,1817,1986,1990, 7008, 7012, 7046, 7062, 7140) showed weak complementation with phage carrying mutations 1049 and 1050 but no complementation with phage carrying mutations 1004 and 1063. Burst assays revealed that these mutations (a total of 30 new mutations) comprised two complementation groups, S, and U, and that mutations 1049 and 1050 were in U while 1004 and 1063 were in S. Representative burst size data presented in Table 8 showed that phage which gave weak complementation in spot tests with phage carrying mutations U1049 and U1050 were defective in S and gave normal bursts in complementation tests with phage carrying U mutations; phage with other S mutations (such as 1004 and 1063) gave bursts only lo-20% of normal when complemented with phage carrying U mutations. The latter low burst sizes are presumably responsible for the lack of complementation observed in the spot tests. DISCUSSION

Approximately 300 new mutants of bacteriophage Mu containing amber mutations in essential genes have been isolated and characterized. Complementation analysis of these mutant strains has led to the removal of cistron 0 and the addition of cistrons T, U, V, W, and Y to the genetic map of Mu (Fig. 1). In general the complementation results were unambiguously positive or negative. When pairs of mutant phage gave normal bursts of 50 to 200 phage per cell from mixed infections, it was concluded that the phage

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TABLE

MU

315

8

COMPLEMENTATIONOF S AND U MUTANT PHAGE~ Burst sizes Mutants

lJ1958

u1050

u1050 u1845 u1958 u7044

3.6 3.0 6.0 2.9 2.7 100 77 58 3.9

2.6 4.9 4.8

u7304 S 1986 s 1294

S7008 None

1.5 1.3

65 85 35 0.7

S7008 21 123

72 92 65 0.02 <.Ol

0.03 0.03

s1004

8

S1063

6.2

12 15 12

10 12

5.4 0.02 0.02 0.04 <.Ol

7.3 4.8 0.01 <.Ol

0.04 <.Ol

H7222 43 77 92 58 123

77 73 68 c.01

None

0.7 0.5 3.9 0.2 0.08 0.01 <.Ol

0.03

a See legend for Table 2.

were defective in different cistrons; when pairs gave the same low burst size as that resulting from single phage infection it was concluded that they were defective in the same cistron. For certain mutant pairs only 3 to 30 phage per cell were produced from mixed infections. Since these burst sizes were lo- to lOOO-fold higher than those produced by infection with each mutant phage alone, it was concluded that these pairs were defective in different cistrons. Mapping experiments (O’Day et al., 1979) revealed that these poorly complementing cistrons were always adjacent to each other. There are a number of possible explanations for the observed low complementation between phage defective in specific pairs of adjacent cistrons. (1) Polarity: Nonsense mutations in promotor-proximal cistrons often cause decreased production of protein from cistrons promotor distal to the mutation; this effect has been termed polarity. In many cases polarity is due to termination of transcription and can be suppressed by mutations in the termination factor, p (Das et al., 1976). The degree of polarity is similar for all promotor-distal cistrons but varies for different nonsense mutations depending on the distance between the nonsense mutation and the next polypeptide initiation site (Newton et al., 1965). This often results in a gradient of polarity within a cistron, with promoter-proximal nonsense

mutations exerting a much stronger polar effect than promoter-distal mutations (Newton et al., 1965). Somewhat different polar effects have been observed for phage A and for RNA phage. Nonsense mutations in late cistrons of A exhibit polarity only on nearby promotor-distal cistrons (Parkinson, 1968) despite the fact that more distant promotordistal cistrons are transcribed from the same promotor (Herskowitz and Signer, 1970). It may be that expression of these cistrons is modulated by some form of translational control and/or messenger RNA processing and that these control mechanisms limit the extent of polarity observed (Ray and Pearson, 1974, 1975). In the case of RNA phage f2 the polar effect of nonsense mutations early in the coat protein cistron is due to the secondary structure of the untranslated RNA which does not allow initiation of the replicase protein (Lodish, 1968, 1970). (2) Intragenic complementation: In the case of the enzyme p-galactosidase, an cABClysDEHFGlTJKLMYNPQVWRSU

FIG. 1. Genetic map of Mu. Capital letters A through U represent cistrons essential for Mu development. The small letter c on the left represents the cistron encoding Mu immunity. The order of cistrons was determined by O’Day et al. (1979) and is presented in the standard orientation with the immunity cistron at the left end.

316

HOWE,

O’DAY,

unusual intragenic complementation is observed with certain pairs of nonsense mutations (Newton, 1969). This complementation occurs by the formation of a partially active P-galactosidase complex from two partial polypeptide chains (Ullmann and Perrin, 1970). One chain results from premature termination of protein synthesis at the nonsense mutation, while the second results from reinitiation of protein synthesis within the cistron at sites naturally resembling protein synthesis initiation sites (Michels and Zipser, 1969). The degree of complementation observed varies greatly for different pairs of nonsense mutations and usually results in a heat-labile activity which is present in less than the normal amount. If this mechanism were causing the low level complementation observed for certain pairs of mutant phage, it would mean that the poorly complementing mutations actually comprise a single cistron rather than two cistrons as designated in this report. It seems unlikely that this mechanism applies to all the poorly complementing cistron pairs because for some cistron pairs the degree of complementation does not vary significantly for phage pairs containing many different nonsense mutations. This mechanism must be considered, however, for those cistron pairs with few mutations and for cistron I, which shows unusual complementation properties. (3) Protein complexing: In this model we postulate that the proteins made by certain pairs of adjacent Mu cistrons form complexes with each other to carry out their functions and that there is preferential complexing of the proteins made from the same phage DNA molecule. In a mixed infection with phage containing nonsense mutations in two such adjacent cistrons, defective complexes would be formed between the prematurely terminated nonsense fragments and the intact proteins of the adjacent cistron. Low level protein mixing might result in the formation of small amounts of normal complexes which could then function to produce the small numbers of phage observed. Although the lack of great variability in complementation ef-

AND SCHULTZ

ficiency with different pairs of mutations may argue against this model, the existing evidence does not rule it out. (4) Overlapping cistrons: In phage 1$X174 some mutations in one cistron affect the activity of the protein produced by a second cistron because the cistrons overlap, i.e., the same nucleotide sequence is read in two different reading frames to produce the two proteins (Brown and Smith, 1977; Smith et al., 1977; Weisbeek et al., 1977). It seems unlikely that overlapping cistrons are the cause for the low level complementation observed in Mu. To explain the poor complementation by this mechanism one would have to postulate that an amber mutation in one cistron resulted in the production of a partially defective protein from the second cistron. In this case the amber mutants should still be partially defective in an Su+ strain. The model further predicts that different amber mutations in one cistron should vary greatly in their effect on the second cistron. Neither of these predictions is substantiated by the data for Mu. The amber mutant strains grow quite normally in an Su+ strain, and there is very little variability in the degree of complementation with different amber mutant phage. The simplest explanation for the poor complementation observed between phage defective in certain adjacent cistrons of Mu would be that the nonsense mutation in the promotor-proximal cistron exerts a polar effect on expression of the promotor-distal cistron. Since transcription of essential genes of Mu occurs from left to right on the genetic map (Fig. 1) (Wijffelman and van de Putte, 1974) such polar effects would be exerted onto the right cistron by nonsense mutations in the left cistron of each pair. One way to determine whether polarity is involved would be to assay complementation under conditions where polarity should not be observed. One such assay would involve measuring complementation of amber mutant phage in a host containing a defective p factor. Unfortunately, this test would not be conclusive because Mu growth is inhibited in such hosts (Howe, unpublished observations). Other experiments involving the

FIVE

NEW CISTRONS

IN BACTERIOPHAGE

complementation analysis of phage with temperature-sensitive mutations, which do not exhibit polar effects, are still in progress. In the absence of direct tests for polarity it may be useful to consider whether the complementation results exhibit a gradient of complementation which would be consistent with a gradient of polarity within a cistron. Cistron pairs H-F and J-K do exhibit such a gradient of complementation. Phage containing the leftmost mutations in H or J show less complementation (more polarity) with F-defective and K-defective phage, respectively, than do phage carrying more rightward mutations in those cistrons. In contrast, complementation results show no gradient for cistron pairs D-E, Y-N, Q-V, and W-R. In the cases of cistrons D, Q, and W the lack of a gradient might be attributable to small distances between mutations; however, for cistron Y the mutations tested were in five deletion groups and should have allowed detection of a gradient if it were present. For cistrons S and U there is a gradient of complementation. Phage carrying the leftmost mutations in S show less complementation with Udefective phage than phage carrying more rightward mutations in S. Although these cistrons are in the invertible G segment, it appears that transcription does occur in the direction from S to U since it occurs from left to right within G when the G segment is in the lytic orientation (C. Wijffelman, personal communication) with a cistron order R S U (Howe et al., 1979). Therefore the gradient observed would be consistent with the hypothesis that nonsense mutations in S are polar on cistron U. This hypothesis was not supported, however, by results of experiments in which complementation of S- and U-defective phage was not increased when the mixed infection was done in an Su+ host which could not suppress the mutant functions but should have suppressed the polar effect (Howe, unpublished observations). For cistron I the interpretation is more complex because of the existence of three groups of mutations. Phage with mutations located at either end of the cistron complement poorly with each other, while

MU

317

phage containing mutations located in the middle of the cistron show no complementation with any I mutant phage. This complementation pattern could be explained by assuming that the mutations comprise a single cistron and that the nonsense and restart polypeptides produced by phage with mutations in the end groups are able to form partially active complexes. The lack of complement&ion by phage with mutations in the middle group would be due to the formation of polypeptide fragments which were too short to interact to produce stable active complexes or possibly also to the lack of formation of a restart fragment. The complementation pattern could also be explained by assuming that the end groups comprise different cistrons and that the chain termination caused by the middle mutations alters the secondary structure of the RNA resulting in faulty processing or accelerated degradation of the RNA or an inability of ribosomes to initiate at the second cistron. Reversion studies have ruled out the possibility that phage in the middle group contain double mutations. Further analysis of complementation properties of phage with temperature-sensitive mutations and identification of protein products affected by the nonsense mutations should provide an explanation for the complementation properties. The reduced complementation of phage carrying B5175 and B5176 mutations with phage carrying A mutations cannot be explained by polarity of the A mutations. Phage with other B mutations located on either side of B5175 and B5176 show essentially normal complementation with phage carrying the same A mutations. It is unlikely that the B5175 and B5176 mutations cause a nonspecific “poisoning” of the cell or prevention of phage development, because phage carrying these mutations complement normally with phage carrying R mutations. Phage carrying these B mutations do not contain a second mutation in the A cistron; the amber mutant phage revert to amber+ at a frequency of -lo-VPFU characteristic of single mutations, and the mutant phage can rescue wild-type growth characteristics from a prophage

318

HOWE, O’DAY, AND SCHULTZ

deletion strain KMBL1644 which is deleted for Mu immunity, cistron A, and part of cistron B (Wijffelman et al., 1973; Howe, unpublished observations). Perhaps the particular nonsense fragment produced by these mutant phage interacts strongly with the A protein made by them and results in the production of a defective protein complex. Now that over 500 total amber mutations in essential cistrons of Mu have been analyzed, we may ask whether all of the essential cistrons of Mu have been discovered. There are several reasons why additional essential cistrons might exist. First, there are three cistrons defmed by only two (7’) and three (C and Q ) mutations; therefore, there may be other cistrons for which no mutations have been found. Second, the majority of amber mutant phage were originally detected using mixed Su+/Su- indicators. Although plating conditions were varied so that a wide variety of mutations would be detected, it is possible that certain types of mutations might not be detectable using this technique. Third, the majority of mutations examined have been amber mutations. If a gene did not contain a sequence able to mutate to amber and also produce active protein when suppressed by the suppressors tested, it would not be detected. Therefore, although it is likely that most essential Mu cistrons are represented in this study, we cannot rule out the possibility that one or a few additional essential cistrons may exist. ACKNOWLEDGMENTS This work was supported by the College of Agricultural and Life Sciences and the Graduate School, University of Wisconsin, Madison, and by NSF Grant PCM75-02465, NIH Grant AI 12731, and ACS Grant UW-IN-350-l to M. M. H. REFERENCES ABELSON, J., BORAM, W., BUKHARI, A. I., FAELEN, M., HOWE, M., METLAY, M., TAYLOR, A. L., TOUSSAINT, A., VAN DE PUTTE, P., WESTMAAS, G. C., and WIJFFELMAN, C. A. (1973). Summary of the genetic mapping of prophage Mu. ViTology 54, 90-92.

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FOWLER, R. G., DEGNEN, G. E., and Cox, E. C. (1974). Mutational specificity of a conditional Escher-ichia coli mutator, m&D 5. Mol. Gen. Genet. 133, 179-191. HENDRIX, R. W. (1971). Identification of proteins coded in phage lambda. In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 355-370. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. HERSKOWITZ, I., and SIGNER, E. (1970). A site essential for expression of all late genes in bacteriophage h. J. Mol. Biol. 47, 545-556. HOWE, M. M. (1973a). Prophage deletion mapping of bacteriophage Mu-l. Virology 54, 93-101. HOWE, M. M. (1973b). Transduction by bacteriophage Mu. Virology 55, 103-117. HOWE, M. M., SCHUMM,J. W., and TAYLOR, A. L. (1979). The S and U genes of bacteriophage Mu are located in the invertible G segment of Mu DNA. Virology 92, 10% 124. LODISH, H. F. (1968). Bacteriophage f2 RNA: Control of translation and gene order. Nature (London) 220, 345-350.

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MICHELS, C. A., and ZIPSER, D. (1969). Mapping of polypeptide reinitiation sites within the P-galactosidase structural gene. J. Mol. Biol. 41, 341-347. NEWTON, A. (1969). Re-initiation of polypeptide synthesis and polarity in the lac operon of Escherichia coli. J. Mol. Biol. 41, 329-339.

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NEWTON, A., BECKWITH, J. R., ZIPSER, D., and BRENNER, S. (1965). Nonsense mutations and polarity in the lac operon of E. coli. J. Mol. Biol. 14, 290-295.

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RAY, P. N., and PEARSON,M. L. (1975). Functional inactivation of bacteriophage A morphogenetic gene mRNA. Nature (London) 253, 647-650. RAZZAKI, T., and BUKHARI, A. I. (1975). Events following prophage Mu induction. J. Bacterial. 122, 437-442.

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SMITH, M., BROWN, N. L., AIR, G. M., BARRETT, B. G., COULSON,A. R., HUTCHISON,III, C. A., and SANGER,F. (1977). DNA sequence at the C termini of the overlapping genes A and B in bacteriophage 1#~X174. Nature (London) 265, 702-705. TESSMAN, I., PODDAR,R. K., and KUMAR, S. (1964). Identification of the altered bases in mutated singlestranded DNA. J. Mol. Biol. 9, 353-363. ULLMANN, A., and PERRIN, D. (1970). Complementation in /3-galactosidase. In “The Lactose Operon” (J. R. Beckwith and D. Zipser, eds.), pp. 143-172. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. VAN VLIET, F., COUTURIER, M., DESMET, L., FAELEN, M., and TOUSSAINT, A. (1978). Virulent mutants of temperate phage Mu-l. Mol. Gen. Genet. 160, 195-202.

WEISBEEK, P. J., BORRIAS, W. E., LANGEVELD, S. A., BASS, P. D., and VAN ARKEL, G. A. (1977). Bacteriophage 4X174: Gene A overlaps gene B. Proc. Nat. Acad. Sci. USA 74, 2504-2508.

WIJFFELMAN, C., and VAN DE PUTTE, P. (1974). Transcription of bacteriophage Mu. Mol. Gem. Genet. 135, 327-337.

WIJFFELMAN, C. A., WESTMAAS,G. C., and VAN DE PUTTE, P. (1973). Similarity of vegetative map and prophage map of bacteriophage Mu-l. Virology 54, 125- 134. ZELDIS, J. B., BUKHARI, A. I., and ZIPSER, D. (1973). Orientation of prophage Mu. Virology 55,289-294.